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Originally published online as doi:10.1189/jlb.0205120 on June 3, 2005

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(Journal of Leukocyte Biology. 2005;78:345-351.)
© 2005 by Society for Leukocyte Biology

Professional phagocytic granulocytes of the bony fish gilthead seabream display functional adaptation to testicular microenvironment

Elena Chaves-Pozo, Victoriano Mulero1, José Meseguer and Alfonsa García Ayala

Department of Cell Biology, Faculty of Biology, University of Murcia, Spain

1 Correspondence: Department of Cell Biology, Faculty of Biology, University of Murcia, Campus Universitario de Espinardo, 30100 Murcia, Spain. E-mail: vmulero{at}um.es


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ABSTRACT
 
It has been shown previously that professional phagocytic granulocytes are present in the testis of the gilthead seabream, a seasonal breeding teleost that offers an excellent model for studying the testicular regression process that occurs in seasonal testicular involution and sex change. It is unexpected that testicular granulocytes produce interleukin-1ß, a regulator for spermatogonia proliferation in mammals, but are not involved in the elimination of degenerative germ cells. Here, we show that phagocytosis and reactive oxygen intermediate (ROI) production were suppressed dramatically in testicular phagocytic granulocytes, compared with their level of activity in the head-kidney, the main hematopoietic organ in fish. Furthermore, testicular-conditioned media modulated migration, phagocytosis, and ROI production of head-kidney phagocytic granulocytes, and the addition of testicular cells impaired their ROI production capacity. Until now, monocytes/macrophages were believed to be the only innate immune cells able to develop into functional subsets, whereas neutrophils only infiltrate the tissues upon infection or inflammation. Our findings demonstrate, however, that fish professional phagocytic granulocytes also display functional adaptation to different microenvironments and strongly suggest a role for these cells in the reorganization of the testis during post-spawning.

Key Words: neutrophils • phagocytosis • testis • reproductive immunology • evolution


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INTRODUCTION
 
Monocytes selectively home to different tissues, presumably under the influence of tissue-specific homing factors, before developing into macrophages that display tissue-specific, functional patterns [1 ]. After entering the tissue, the monocyte/macrophage migrates to the tissue parenchyma, an environment that significantly influences the function of macrophages in such a way that macrophages resident in different tissues display different patterns of function [1 ]. However, no comparable behavior has been found in other professional phagocytes, such as neutrophils [2 ]. The term "neutrophils" reflects the light microscopic appearance of human cells after staining, but it is less appropriate for the functional equivalent of human neutrophils in other animal species. In this respect, fish granulocytes have long been a source of confusion as a result of the wide morphological differences between major teleost groups [3 , 4 ]. It is important that the staining properties of the cells should not be taken as a direct reference to their function in host defenses. The acidophilic granulocytes of the teleost gilthead seabream, despite their staining pattern, display similar functions to human neutrophils. For example, they are the most abundant circulating granulocytes and are recruited rapidly from the head-kidney, the main hematopoietic organ in fish, to the infection site [5 ]. In addition, their main role is the phagocytosis of bacteria, which is accomplished by coordinating their attachment and internalization to the release of reactive oxygen intermediates (ROIs) into the phagocytic vacuole [5 6 7 ]. Bearing all this in mind, henceforth, we will use the term "neutrophil-like cells" (sbNLCs) to refer to the professional phagocytic granulocytes of the gilthead seabream.

Previous results have demonstrated that sbNLCs are present in the testis of the gilthead seabream [8 ], a seasonal breeding teleost that is an excellent model for studying the testicular regression process, which occurs in seasonal testicular involution and sex reversion. In contrast to sbNLCs from the head-kidney, testicular sbNLCs constitutively produce interleukin-1ß (IL-1ß), a potent growth factor for mammalian spermatogonia and Leydig cells [9 10 11 ], rather than being involved in the phagocytosis of degenerative germ cells [8 , 12 ]. In addition, sexual hormones influence the production of ROIs and IL-1ß by head-kidney sbNLCs in vitro [8 ]. These results, together with the fact that sbNLCs do not proliferate in the testis [8 ], suggest that they are recruited from the head-kidney and that testis-specific microenvironment factors might influence their activities. Here, we show that the phagocytic and ROI production capacities of testicular sbNLCs are suppressed dramatically. Furthermore, testicular conditioned media (TCM) influence the migration, phagocytosis, and ROI production of head-kidney sbNLCs, and the presence of testicular cells strongly inhibits their capacity to produce ROIs. These findings demonstrate that fish professional phagocytic granulocytes may display functional adaptation to different microenvironments and strongly suggest an unexpected role for these cells other than immune surveillance.


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MATERIALS AND METHODS
 
Animals, cell suspensions, and TCM
Healthy specimens of mature, male gilthead seabream (Sparus aurata L.), with body weights of 100–200 g, were obtained from Culmarex S.A. (Murcia, Spain). Specimens were decapitated, and the gonads and the head-kidneys were removed in different stages of the reproductive cycle: spermatogenesis, spawning, post-spawning, and resting stages. The gonadosomatic index (GSI) was calculated as 100 x (Wg/Wf), where Wg is the gonad weight, and Wf is the body weight. Gonads and head-kidneys were dissociated as described elsewhere [8 , 13 ]. Some gonads were processed by light microscopy and immunostained with a monoclonal antibody (mAb) specific to sbNLCs (G7) [6 ], as described [8 ]. Testicular pieces were cultured for 24 h in sbt-L15 medium [13 ] with shaking and optimal oxygen availability before TCM was collected, centrifuged to discard the cells, and stored at –80°C.

Magnetic-activating cell sorting (MACS)
Head-kidney or testicular cell suspensions were incubated with a 1:10 dilution of G7 mAb, washed twice with phosphate-buffered saline (PBS) with 2 mM EDTA (Sigma Chemical Co., St. Louis, MO) and 5% fetal bovine serum (Gibco, Grand Island, NY), and then incubated with 100 µl per 107 cells micromagnetic bead-conjugated anti-mouse immunoglobulin G antibody (Miltenyi Biotec, Auburn, CA). After washing, G7+ cells were collected by MACS following the manufacturer’s instructions. Aliquots of 105 cells per fraction were then incubated with 1:100 fluorescein isothiocyanate (FITC)-labeled anti-mouse F(ab')2 fragments of goat antibody (Sigma Chemical Co.) to determine the purity of the fractions.

Cell treatments
Aliquots of 5 x 105 head-kidney cells or head-kidney G7+ cell-enriched fractions (hkG7+) were incubated with 0, 0.1, 10, 25, and 50% of TCM in sbt-L15 medium for 4, 20, and 46 h or with 5 x 105 testicular cells for 1 h.

Phagocytosis assay
Aliquots of 5 x 105 testicular cells, testicular G7+ cell-enriched fractions (tG7+), head-kidney cells, or hkG7+ preincubated with TCM were challenged with FITC (Sigma Chemical Co.)-labeled Vibrio anguillarum (strain R-82, serotype 01) for 60 min as described [13 ]. When needed, the phagocytosis was stopped in ice-cold PBS, and the cells were immunostained with the G7 mAb and analyzed by flow cytometry [13 ].

ROI production assay
The production of ROIs by head-kidney cells or hkG7+ preincubated with TCM triggered by phorbol myristate acetate (PMA; Sigma Chemical Co.) and/or formalin-killed V. anguillarum (1:20) was measured as the luminol-dependent chemiluminescence [14 ]. The production of ROIs by testicular cells, tG7+, or head-kidney cells triggered by PMA was measured as the dihydrorhodamine 1,2,3-dependent fluorescence, using flow cytometry [15 ].

Migration assay
The migratory activity of head-kidney cells was assessed using a 48-well microchemotaxis chamber (Neuro Probe, Gaithersburg, MD) as described [16 ]. To the lower well of the chamber, 0, 0.1, 10, 25, and 50% of TCM, 1:10 diluted gilthead seabream serum as a positive control, or medium alone as a negative control were added.

Viability assay
Aliquots of head-kidney cells preincubated with TCM were diluted in 200 µl PBS containing 40 µg/ml propidium iodide. The number of red fluorescent cells (dead cells) from triplicate samples was analyzed by flow cytometry.

Statistical analysis
Data were analyzed by one-way or two-way ANOVA and unpaired Student’s t-test to determine differences between groups.


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RESULTS
 
sbNLCs are actively recruited to the testis
We used a mAb, G7, which is specific to sbNLCs [5 ], to determine, by flow cytometry, the percentage of these cells in the testis of the gilthead seabream throughout its reproductive cycle. The results show that approximately 80% of the granulated cells (R2 region) of the testis were sbNLCs (G7+ cells; Fig. 1a 1b 1c ). Moreover, these cells increased in number sharply (Fig. 2a ) and appeared close to germ cells (Fig. 2b) after shedding of the spermatozoa (post-spawning). The change in G7+ cell percentages during the reproductive cycle together with the fact that they do not proliferate in the testis [8 ] suggest that their recruitment is regulated by the physiological status of the testis. In the gilthead seabream, the head-kidney is the main hematopoietic organ, and it actively responds to the sbNLC requirements during infection [5 ]. This led us to examine whether TCM would induce head-kidney cell migration. The staining pattern and morphological features of the migrating cells confirmed that testicular soluble factors were able to recruit sbNLCs (data not shown). Head-kidney sbNLCs, particularly, migrated toward TCM in a dose-dependent manner, the highest response being observed when 10% and 25% of TCM were used (Fig. 2c) .



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  		Figure 1. Flow cytometry analysis of the phagocytic activity of testicular (a–c) and head-kidney (d–f) cells. Representative dot-plot of forward scatter (FSC) versus side-scatter (SSC; a, d) and green versus red fluorescence of cells from gate R2 (b, c, e, f). Control cells (b, e). Cells challenged with FITC-labeled V. anguillarum (green fluorescence) and then immunostained with the G7 mAb (red fluorescence; c, f).



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  		Figure 2. Quantification and localization of testicular sbNLCs and migration of head-kidney sbNLCs to TCM. (a) The GSI versus the percentage of testicular sbNLCs (n=7–19 fish/month). Testicular cell suspensions were immunostained with the G7 mAb and then analyzed by flow cytometry. D, December; J, January; F, February; M, March; A, April; MY, May; Jn, June; Jl, July. (b) Testicular sbNLCs (arrows) located in the germinal epithelium were immunostained with the G7 mAb. Original magnification, x100. (c) Number of migrating head-kidney sbNLCs per field. The results are presented as mean ± SE from a representative experiment performed in triplicate. Versus control cells: *, P ≤ 0.05.

Testicular sbNLCs show impaired phagocytic activities
To check whether the sbNLCs might have similar roles in the testis as in other immune tissues, we examined their main functional activities (phagocytosis and ROI production) and found that as strikingly little as 1% of the testicular sbNLCs phagocytosed the bacterium V. anguillarum in all the stages of the reproductive cycle (Fig. 1c and data not shown). In sharp contrast, approximately 75% of the head-kidney sbNLCs were able to phagocytose this bacterium (Fig. 1f) . In the same way, many fewer testicular-granulated cells (R2 region) produced ROIs (Fig. 3a ) and showed a response that was eightfold lower in intensity than their head-kidney counterparts (Fig. 3b) . To further confirm the contribution of the sbNLCs to ROI production, we used MACS to obtain testicular sbNLC-enriched fractions (tG7+). Using these tG7+ fractions, which had approximately threefold more sbNLCs than the entire testicular cell suspension (Fig. 4a and 4b ), we confirmed that testicular sbNLCs displayed much lower phagocytic activity (Fig. 4c) . It is interesting that the ROI production assay using tG7+ cells showed that the majority of the ROI-producing cells in the testis was sbNLCs (Fig. 4d) , although the percentage of cells that responded was low.



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  		Figure 3. Flow cytometry analysis of the production of ROIs by testicular and head-kidney cells. (a) Percentage of ROI-producing cells and (b) the intensity of the response [mean fluorescence intensity (MFI)] in both tissues. The results are presented as mean ± SE from a representative experiment performed in triplicate. Versus head-kidney cells: *, P≤ 0.05.



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  		Figure 4. Flow cytometry analysis of the production of ROIs by testicular cells after immunopurification by MACS. Representative dot-plots of G7+ cells from whole testicular cell suspensions (a) and testicular sbNLC-enriched fractions (tG7+; b) were immunostained with the G7 mAb. The percentage of cells from whole testicular cell suspensions and tG7+ able to phagocytose FITC-labeled V. anguillarum (c) and to produce ROI upon stimulation with 10 ng/ml PMA (d) is shown. The results are presented as mean ± SE from a representative experiment performed in triplicate. Versus whole testicular cell suspensions: *, P ≤ 0.05.

The testicular environment inhibits the phagocytic activities of head-kidney sbNLCs
As all our previous data suggested that testicular sbNLCs are originated in the head-kidney, we next hypothesized that head-kidney leukocyte functions might be modulated by testicular soluble factors or cells. Exposure of head-kidney cells to low concentrations (0.1% or 1%) of TCM for 20 h slightly increased the phagocytic activity (Fig. 5a ) and ROI production (Fig. 5b) . However, longer exposures or higher concentrations inhibited both activities. Thus, 46 h exposure to TCM concentrations higher than 10% decreased the phagocytic activity (Fig. 5a) , and exposure to 50% of TCM decreased the ROI production at all the time-points assayed (Fig. 5b) . It is important to note that cell viability was unaffected by the treatments and ranged from 86 ± 2 to 90 ± 2, as assayed by propidium iodide staining. To further confirm whether the head-kidney sbNLC activities are influenced by tissue-specific factors, we obtained head-kidney sbNLC-enriched fractions (hkG7+) with 95% of purity (three to fourfold enrichment; data not shown). The hkG7+ showed a rather weak inhibition of ROI production after 20 h of culture with 1–10% of TCM but a significant increase after 20 and 46 h with the highest concentration (50%; Fig. 5c ). Therefore, isolated head-kidney sbNLCs seem to be more sensitive to TCM than when they are in the presence of other head-kidney immune cells (Fig. 5c vs. 5b ).



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  		Figure 5. Phagocytosis and ROI production of head-kidney cells preincubated with TCM. Whole head-kidney cell suspensions (hk; a, b) or head-kidney sbNLC-enriched fractions (hkG7+; c) were incubated for different times with the indicated dilutions of TCM, and then the phagocytosis of FITC-labeled V. anguillarum was assayed by flow cytometry (a), and the production of ROIs triggered by PMA was assayed by chemiluminiscence (b, c). Horizontal lines represent the control value (cells incubated with medium alone). The results are presented as mean ± SE from a representative experiment performed in triplicate. Versus control cells: *, P ≤ 0.05.

The above results prompted us to examine whether the inhibition of the phagocytic activities of testicular sbNLCs may also depend on cell-cell interactions in the testis. The results show that preincubation of head-kidney leukocytes with testicular cells for 1 h resulted in a strong inhibition of the production of ROIs triggered by V. anguillarum (Fig. 6 ). Thus, the head-kidney cells preincubated with testicular cells and then challenged with the bacterium showed a 4.5-fold or 12-fold decrease in ROI production compared with the activity of nonchallenged or challenged head-kidney cells, respectively (Fig. 6) .



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  		Figure 6. ROI production of head-kidney cells preincubated with testicular cells. Production of ROIs by head-kidney cells alone (hk) or in the presence of testicular cells (hk+testis) for 1 h and then challenged (hk+vibrio and hk+testis+vibrio) or not with V. anguillarum. RLU, Relative light units. The results are presented as mean ± SE from a representative experiment performed in triplicate. Versus nonchallenged cells: *, P ≤ 0.05.


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DISCUSSION
 
In teleost fish, the number of reported, identifiable myeloid lineages and their cellular morphologies differs between species [17 ], as it does among mammalian species [18 ]. Distinct neutrophilic, eosinophilic, and basophilic granulocytes have been morphologically identified in fish, but this does not necessarily mean that these cell types play similar roles as in mammals [19 ]. In the bony fish gilthead seabream, the acidophilic granulocytes display similar functions to human neutrophils despite their opposite staining pattern. In short, they are recruited rapidly to the site of inflammation [5 ] and are highly specialized to attach, internalize, and kill bacteria by the production of ROIs [5 , 6 ]. Taking this into account and to avoid confusing this cell type and other granulocytes, we have used the term sbNLCs to refer to the professional phagocytic granulocytes of the gilthead seabream.

The monocyte/macrophage system is characterized by its functional plasticity and ability to adapt to tissue-specific microenvironments displaying tissue-specific, functional patterns. In spite of this functional plasticity, the cells are thought to be involved in the phagocytosis of host and foreign particles, as well as in immune regulatory functions [20 ]. The data presented in this study strongly suggest that mobile sbNLCs are also able to specifically home a tissue, i.e., the testis, in response to a physiological need. Moreover, such behavior on the part of sbNLCs might be extended to other tissues, as the peritoneal exudate of noninfected fish contains ~20% of resident sbNLCs [12 ]. In mammals, it has been reported that the uterine cycle involves the recruitment and infiltration of large numbers of circulating eosinophils at estrus [21 ] and that cytokines are involved in such a process [22 ]. These observations show some similarities with the infiltration of sbNLCs in the testis, as the fish testis and the mammalian uterus show cyclical changes. However, the cellular mechanisms and the functions of this uterine phenomenon also remain poorly understood. In fact, mice devoid of eosinophils show normal estrous cycles and reproductive functions [22 , 23 ].

One of the most important findings of this study is that testicular sbNLCs show impaired phagocytic and ROI production activities compared with their head-kidney counterparts, although they are the majority of cells able to produce ROIs in the testis upon PMA stimulation. These data demonstrate that the activities of sbNLCs are modified by the microenvironment of the testis. In fact, testicular sbNLCs are able to constitutively produce IL-1ß [8 ], whereas head-kidney, peripheral blood, and peritoneal exudate sbNLCs only produce IL-1ß upon activation [12 ]. Furthermore, the phagocytic activities of head-kidney sbNLC are modulated by testicular soluble factors or cells, suggesting the functional plasticity of sbNLCs. It is worthy of mention that isolated head-kidney sbNLCs (G7+ cells) respond better to TCM than whole head-kidney cell suspensions, suggesting that sbNLC activities are also influenced by other immune cells. Notably, the presence of testicular cells dramatically inhibits the production of ROIs by sbNLCs. Although the identity of the molecules and receptors mediating this effect requires further studies, it is tempting to speculate that the phagocytic activity of sbNLC is down-regulated in the testis to avoid the elimination/damage of germ cells. We believe that the illumination of the mechanisms orchestrating the inhibition of sbNLCs in the testis might help to further understand the privileged immune status of the testis and even the development of human autoimmune diseases.

In regard to the functional differences between gilthead seabream immune and testicular sbNLCs, it is possible to establish several similarities with the testicular monocyte/macrophage system of mammals. Thus, mammalian testicular macrophages display a novel cytokine secretion profile compared with peritoneal macrophages [24 ] and retain their cytotoxic and phagocytic capacities, although they have greatly diminished proinflammatory functions and exhibit immunosuppressive activity [25 ]. Moreover, the recruitment and maintenance of the resident macrophages are clearly under the control of testicular soluble factors [26 ], such as macrophage migration inhibitory factor [27 ] and monocyte chemoattractant protein-1 [28 , 29 ]. The testicular sbNLCs are also recruited by testicular soluble factors, show a different cytokine production profile [8 ], and retain some phagocytic and ROI production capabilities, although they are impaired severely by the testicular microenvironment. Approximately 450 million years of evolutionary divergence separate mammals and bony fish, providing considerable scope for divergence in cellular and morphological features of myeloid lineage [30 ]. Although the essential features of a multilineage myeloid system for host defense are conserved [4 ], little is known about the roles and the cells involved in immune-endocrine interactions in peripheral tissues in fish. Although a monocyte/macrophage system involved in immune-endocrine interaction cannot be ruled out, our data demonstrate that a neutrophil-like cell migrates in response to specific stimuli and displays a functional plasticity, which is likely influenced by soluble factors and cell interactions in the testis. This suggests that the differentiation between the different myeloid lineages is not as well established in lower vertebrates as it is in mammals.


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ACKNOWLEDGEMENTS
 
This work was supported by the Spanish Ministry of Education and Science (Grants BIO2001-2324-C02-02, AGL2002-03529, and AGL2002-04306-C03-01) and Fundación Séneca (Grant PI-51/00782/FS/01 and fellowship to E. C-P.). We thank the "Servicio de Apoyo a las Ciencias Experimentales" (S.A.C.E.) of the University of Murcia for assistance with cell culture, Drs. A. E. Toranzo and J. L. Barja for the bacterium, and Dr. S. González for her critical reading of the manuscript.

Received February 28, 2005; revised April 19, 2005; accepted May 3, 2005.


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E. Chaves-Pozo, S. Liarte, L. Vargas-Chacoff, A. Garcia-Lopez, V. Mulero, J. Meseguer, J.M. Mancera, and A. Garcia-Ayala
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B. Lutton and I. Callard
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